Construction of an eGFP expression plasmid under control of T7 promoter and IRES sequence for assay of T7 RNA polymerase activity in mammalian cell lines

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site [IRES] sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics

Comparative molecular PCR-RFLP study of native herpes simplex virus type 1 [HSV-1] with KOS strain

Recent research on several DNA fragments covering open reading frames [ORF] 1-37 shows a new genetic marker in ORF 6 which is specific for differentiating wild-type varicella-zoster virus [VZV] strains from Oka varicella vaccine strain. On the other hand, herpes simplex virus [HSV] genome analysis by restriction enzymes is used to differentiate types one and two of the virus and even strains of each type. Previous studies using PCR-sequencing technique have shown that the thymidine kinase [TK] gene of HSV-1 is polymorphic. In this study, TK gene and DNA binding protein [UL29] gene of HSV-1 were selected. Both genes were analyzed with restriction endonucleases in order to identify a genetic marker for differentiating native strains of HSV-1 from the foreign strain. Three isolates of HSV-1 as well as standard strain of KOS were propagated in Vero cells. Initially, a pair of specific primers for each gene was designed to amplify UL29 and TK genes of these isolates. Subsequently, PCR products of these genes were digested separately with five restriction enzymes and subjected to polyacrylamide gel electrophoresis. Using PCR-RFLP [Restriction fragment length polymorphisms] technique, results indicate that the patterns of restriction endonuclease digestion of UL29 and TK genes of the three isolates show no differences when compared to KOS strain. The genotypes of Iranian isolates are the same as KOS genotype and both genotypes are derived from a common ancestor. Hence, it can be postulated that in the process of random population flow among Iran, Europe and USA, the original KOS strain infected the Iranian population at some point in time